Support is requested in this competitive renewal to continue our studies of mechanisms of acquired drug resistance. We propose a comprehensive study of the kinds of changes in protein abundance and structures that occur in cancer cells when they become resistant to different chemotherapeutic agents. Mass spectrometry-based proteomics strategies will be used to compare the abundances and structures of proteins in MCF-7 human breast cancer cells with three sublines selected by exposure to etoposide, mitoxantrone and melphalan. It is expected that a much larger array of proteins will be found to be altered than has been previously recognized. We also expect that thoughtful consideration of the kinds of changes observed will provide new insights into mechanisms of acquired resistance, and into the hyper-sensitivity of transformed cells. New therapeutic targets may be identified. This is not envisioned as a high throughput project, but rather, overlapping and reiterative comparisons of organelles and sub-fractions will be made to allow structural changes due to mutations or post-translational modifications to be recognized and elucidated. In a parallel and related investigation, patterns of protein alkylation by radio-isotope labeled melphalan will be compared in susceptible and resistant cells, sub- fractions and organelles. Correlations, or the lack thereof, will be sought between the nature of the alkytation targets, changes in the alkylation targets, and changes in cell biochemistry defined in the comparative study.